A sensitive subject: Haploinsufficiency of the yeast VMA13 gene in simple and compound heterozygotes

Vacuolar-proton translocating-ATPases (V-ATPases) are the membranebound transporters responsible for controlling pH levels in intracellular compartments. The V-ATPase enzyme is a multi-subunit complex consisting of two distinct domains, a cytosolic V1 region and a membrane-bound Vo domain. While the interface between V1 and Vo is not as well understood, studies suggest the possibility of multiple peripheral stalks tethering the complex. In response to glucose deprivation, the V1 and Vo sectors of the enzyme are capable of dissociating into separate, and inactive, domains as a means of regulation. At least 4 V1 subunits (C, H, E, and G) are believed to form the peripheral stalk(s) of the V-ATPase. Of considerable interest amongst peripheral stalk subunits has been the H subunit, a protein which has been implicated in the suppression of VATPase activity in free V1 domains and also recently shown to play a key a role in the structural and functional coupling of V1 to Vo. In this study, we’ve identified a clear haploinsufficiency for the VMA13 gene (encoding subunit H). Initially manifested as a growth defect at elevated pH and/or in the presence of extracellular calcium, haploinsufficiency of the VMA13 gene proved to have more profound biological implications on the cell, reducing vacuolar acidification in vivo, ATPase activity at the vacuole, and allowing for ATP hydrolysis in free V1 complexes in the cytoplasm. The importance of VMA13 gene product as both a peripheral stalk protein and structural/functional regulator of the enzyme led us to further investigate the biochemical basis of this haploinsufficiency by selectively forming compound heterozygous mutants. iii After crossing vma13 mutants to various vma knockouts, we found altered phenotypes of compound haploinsufficiencies for the enzyme. Creating an imbalance of both subunits H (Vma13p) and c (Vma3p) exacerbated haploinsufficiency phenotypes of cells, slowing growth on all media, eradicating in vivo acidification at the vacuole, increasing cytosolic ATPase activity, and reducing overall enzyme activity and assembly at the vacuolar membrane. In contrast to these results, altering the balance of both H and B subunits (Vma13p and Vma2p respectively), led to the opposite result. Together, the subunit locations and phenotypes of compound heterozygous vma mutants are explained, at least in part, by their biochemical phenotypes. A perturbation of H subunit stoichioimetry creates a gain in ATPase activity in the stable population of free V1 sectors in the cytoplasm and results in an uncoupling of ATP hydrolysis and proton translocation, in essence wasting away intracellular energy stores. The wasting of energy coincides with the poor growth of vma13Δ heterozygotes and, along the same lines, the level and activity of this active free V1 population underlies many phenotypic differences amongst compound heterozygous mutants. While many compound mutant phenotypes can be explained by this biochemical phenomenon, a reduction of ATPase activity at the vacuole along with reduced V-ATPase assembly at the vacuole for some strains (e.g. vma13Δ x vma3Δ heterozygotes) suggests that perturbation of certain subunit levels may also have synergistic effects on overall enzyme stability and assembly. In summary, this data sheds light onto subunits in the V-ATPase iv enzyme normally present, in vivo, in limited quantities, highlighting their importance for an active and healthy enzyme